The present invention relates to a support comprising oligo- or polynucleotides covalently linked at their 5xe2x80x2- or 3xe2x80x2-termini to at least one major surface of said support through bifunctional spacers and bifunctional linkers, the use of the support according to the invention, the preparation of the support according to the invention, and a method for establishing transcription profiles.
Analyses performed on a molecular-biological level are increasingly gaining importance. In most cases, a mixture of nucleic acids to be analyzed is hybridized with so-called probes and characterized in such methods. Especially for problems in which a large number of polynucleic acids of different kinds are to be detected simultaneously, there are bottlenecks in the method. It is attempted to perform a large number of analytical steps within a short period of time, especially by a parallel operation of the process. There is often a problem in that support systems on which the hybridization experiments can be performed have a limited space capacity. Therefore, it is attempted to solve these problems by using supports on which a large number of samples can be placed. In particular, the prior art describes supports which have micrometer or nanometer compartments for receiving correspondingly small volumes of the analytes which are mostly in solution. Appropriate support systems can be obtained, for example, by etching the surfaces of wafers made of silicon.
E. M. Southern (E. M. Southern et al. (1992), Nucleic Acids Research 20: 1679 to 1684, and E. M. Southern et al. (1997), Nucleic Acids Research 25: 1155 to 1161) describes the preparation of so-called oligonucleotide arrangements by direct synthesis on a glass surface derivatized with 3-glycidoxypropyltrimethoxysilane and then with a glycol.
The publication by S. P. A. Fodor (A. C. Pease et al. (1994), Proc. Natl. Acad. Sci. USA 91: 5022 to 5026) relates to a similar method. The in situ oligonucleotide synthesis described therein is performed by fully automated light-controlled combinatorial chemistry. The direct synthesis of oligonucleotides on a glass support allows a maximum length of about 30 bases. Ensuring a correct course of the synthesis for an individual sequence of longer oligonucleotides, if at all possible, involves an expenditure which is no longer justifiable. As a guide DNA, these oligonucleotides allow the hybridization of only a rather short length of the analyte nucleic acid. To circumvent this drawback, several oligonucleotides are synthesized as guide DNAs for each nucleic acid to be analyzed. This results in higher demands for space and thus a larger sample volume of the analyte nucleic acid. Further, the small length of the oligonucleotides does not preclude cross hybridizations with different analyte nucleic acids. This makes an unambiguous assignment of the recorded signals difficult.
For the preparation of so-called DNA chips, P. O. Brown (DeRisi et al. (1997), Science 278: 680-686) discloses polylysine-coated glass surfaces to which minute DNA quantities are applied dropwise by capillary techniques. However, the immobilization of the guide DNA on a polylysine surface adversely affects hybridization and thus considerably increases the detection limit and reduces the reliability in the detection of the analyte nucleic acids.
L. M. Smith (Z. Guo et al. (1994), Nucleic Acids Research 22: 5456-5465) describes a technology for the immobilization of oligonucleotides in which oligonucleotides are derivatized with a 5xe2x80x2-terminal amino group and then applied to a glass surface derivatized with 3-aminopropyltrimethoxysilane and then with 1,4-phenyldiisothiocyanate. While the chemistry used for the immobilization of the oligonucleotides avoids the drawbacks of the previously described systems, there are still the previously mentioned drawbacks caused by the use of short oligonucleotides as the guide DNA.
Such systems can be prepared only with a high expenditure usually, and often fail to reach a satisfactory capacity of sample compartments which would be necessary for appropriate parallelization.
In addition, the use of complete cDNAs is not advisable. On the one hand, cross-reactions will occur in highly homologous gene families, and on the other hand, the cDNAs contain repetitive elements which may result in non-specific hybridizations. This may result in artifacts. In comparisons between different species, the mentioned artifacts may result in a severe limitation of the method.
Thus, it has been the object of the present invention to provide a support which avoids the mentioned drawbacks of the prior art. In particular, the support according to the invention should be able to bind nucleic acids, preferably having a defined sequence and, if possible, the same length, in high densities and allow a high level of parallelization of samples to be examined.
According to the invention, this object is achieved by a support having the features of claim 1. Preferred embodiments of the support according to the invention are found in the dependent claims. The present invention also relates to a method for the preparation of the support according to the invention and its use. The provision of the support according to the invention enables a novel and inventive method which advantageously enables the quantification of transcription profiles.